Expression and characterization of a Synechocystis PCC 6803 P-type ATPase in E. coli plasma membranes.
In a previous paper, we published the sequence of a P-type ATPase gene from Synechocystis 6803 [Geisler et al. (1993) J. Mol. Biol. 234, 1284] which showed significant homologies to eukaryotic calcium ATPases. To investigate the specificity and activities of this plasma membrane-bound enzyme, we expressed the slightly modified gene in an ATPase deficient E. coli strain. The expressed ATPase showed an apparent molecular mass of about 97kDa and is localized in the E. coli plasma membranes. The introduced 6xHis tag at the N-terminus allowed the purification of the Synechocystis 6xHis-ATPase by single-step affinity chromatography using a Ni2+-nitrilotriacetic acid resin. The ATPase activity of the enzyme is inhibited by vanadate (IC50 = 119 microM), N-ethylmaleimide, N,N-dicyclohexylcarbodiimide, and inhibitors of eukaryotic sarco(endo)plasmic reticulum Ca2+-ATPases; however, it is stimulated by thapsigargin. Formation of phosphorylated enzyme intermediates depends on calcium ions indicating that the Synechocystis P-ATPase acts as a calcium pump equivalent to eukaryotic sarco(endo)plasmic reticulum Ca2+-ATPases.
Geisler
M
M
Institut für Biochemie der Pflanzen, Heinrich-Heine-Universität Düsseldorf, Germany.
Koenen
W
W
Richter
J
J
Schumann
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases